The overall objective of this project is the development of an effective vaccine to prevent invasive, life- threatening infections by the medically important bacterial pathogen Staphylococcus aureus. Because S. aureus cannot always be controlled by antibiotics and methicillin-resistant isolates are becoming increasingly prevalent in the community, a S. aureus vaccine is sorely needed. GlycoVaxyn, a small biotechnology firm in Switzerland, has developed a proprietary technology that enables the manufacture of bioconjugate vaccines based on complex polysaccharide structures. A novel Campylobacter enzyme called PglB is able to transfer an oligosaccharide to a protein consensus sequence, thereby allowing the production of glycoproteins in bacterial cells. This protein glycosylation system has been functionally transferred into Escherichia coli, and it allows the conjugation of an antigenic polysaccharide to a designer protein of choice through an N-glycosidic linkage. Conjugated vaccines have been produced in E. coli by co-expression of PglB, a protein carrier, and an antigenic polysaccharide cluster. In this approach, the conjugated vaccine can be extracted from the periplasm of E. coli and purified. In preliminary studies GlycoVaxyn has prepared a novel vaccine by engineering E. coli to express the S. aureus capsule type 5 polysaccharide (CP5) vaccine covalently linked to the carrier protein EPA (nontoxic Pseudomonas aeruginosa exoprotein A). The vaccine was purified, characterized, and injected into mice and rabbits to demonstrate its immunogenicity. High titered rabbit antibodies to CP5-EPA promoted opsonophagocytic killing of two different S. aureus strains in an in vitro assay with human neutrophils. In addition, passive transfer of CP5-EPA antibodies resulted in a 98% reduction in S. aureus bacteremia in mice. These data provide a strong foundation for the proposed studies to determine whether multicomponent S. aureus bioconjugate vaccines will protect against disease provoked by multiple S. aureus strains in a variety of infection models. Our specific aims are to (1) utilize GlycoVaxyn's proprietary glyco-engineering technology to produce a tetravalent bioconjugate vaccine against S. aureus: CP5-clumping factor A (ClfA) and CP8-alpha toxoid; (2) perform in vitro assays to evaluate the functionality of antibodies elicited by the bioconjugate vaccines; (3) perform active immunization experiments to test the efficacy of the multi-component bioconjugate vaccine in rodent models of staphylococcal infection, including bacteremia, pneumonia, wound infection, and endocarditis; (4) investigate the immune correlates of protection against S. aureus infection by passive immunization and T cell depletion experiments; (5) development of a GMP compatible production process in E. coli for the tetravalent bioconjugate. By glyco-engineering technology, capsular polysaccharides are conjugated to known protective peptides without denaturing the protein carrier, thus conserving B cell epitopes. This approach paves the way for the biosynthesis of previously unattainable vaccine materials in a cost effective manner. The overall goal of this project is the development of an effective vaccine to prevent invasive, life-threatening infections by the medically important bacterial pathogen Staphylococcus aureus. A new technology allows for bacterial synthesis of vaccine proteins coupled to polysaccharides with known vaccine potential. The bioconjugate vaccines will be tested in animal models of staphylococcal infection and processed for initial safety testing in humans.